Format for Individual Target pages (copy this list to new Target page and then fill in for your target):
*Target (protein/gene name):tyrosine hydroxylase *NCBI Gene # or RefSeq#: 21823 *Protein ID (NP or XP #) or Wolbachia#: 033403.1 *Organism (including strain): Toxoplasma gondii Etiologic Risk Group (see link below): Risk group 2 *Background/Disease Information (sort of like the Intro to your Mini Research Write up):
It is a parasitic disease caused by the protozoan Toxoplasma gondii. The parasite infects most genera of warm-blooded animals, including humans, but the primary host is the felid (cat) family. Animals are infected by eating infected meat, by ingestion of feces of a cat that has itself recently been infected, and by transmission from mother to fetus. Cats are the primary source of infection to human hosts, although contact with raw meat, especially lamb, is a more significant source of human infections in some countries. Fecal contamination of hands is a significant risk factor. Link to TDR Targets page (if present): http://tdrtargets.org/targets/view?gene_id=237217 Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) http://www.ncbi.nlm.nih.gov/gene/21823 Essentiality of this protein:
Gene Ontology Function: (mouse over links to read term descriptions) GO:0005506 iron ion binding GO:0004497 monooxygenase activity
Process: (mouse over links to read term descriptions) GO:0055114 oxidation reduction GO:0009072 aromatic amino acid family metabolic process Complex of proteins?: no Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): no chemical compounds *EC#:1.14.16.2 Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.14.16.2
Figure 1- Brenda reaction mechanism of catalysis by 4a-hydroxytetrahydrobiopterin
6,7-Dimethyl-5,6,7,8-Tetrahydropterine 100 mg in glass bottle for $105.00. 1l Tris HCL $27.00, L-Tyrosine Solution 50g for $65.50 Structure Available (PDB or Homology model) PDB # 2MDA Expression Information (has it been expressed in bacterial cells): yes in escherichia coli Purification Method: one of many purification methods is chromatography Image of protein (PyMol with features delineated and shown separately):
Figure 2 -Tyrosine hydroxylase, protein shown as cartoon, colored from the N-terminus to the C-terminus in rainbow
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
*length of your protein in Amino Acids: 527 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 58392.9 Molar Extinction coefficient of your protein at 280 nm wavelength: 46215 M^-1 cm^-1 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
Format for Individual Target pages (copy this list to new Target page and then fill in for your target):
*Target (protein/gene name):tyrosine hydroxylase*NCBI Gene # or RefSeq#: 21823
*Protein ID (NP or XP #) or Wolbachia#: 033403.1
*Organism (including strain): Toxoplasma gondii
Etiologic Risk Group (see link below): Risk group 2
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
It is a parasitic disease caused by the protozoan Toxoplasma gondii. The parasite infects most genera of warm-blooded animals, including humans, but the primary host is the felid (cat) family. Animals are infected by eating infected meat, by ingestion of feces of a cat that has itself recently been infected, and by transmission from mother to fetus. Cats are the primary source of infection to human hosts, although contact with raw meat, especially lamb, is a more significant source of human infections in some countries. Fecal contamination of hands is a significant risk factor.
Link to TDR Targets page (if present):
http://tdrtargets.org/targets/view?gene_id=237217
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
http://www.ncbi.nlm.nih.gov/gene/21823
Essentiality of this protein:
Gene Ontology
Function: (mouse over links to read term descriptions)
GO:0005506 iron ion binding
GO:0004497 monooxygenase activity
Process: (mouse over links to read term descriptions)
GO:0055114 oxidation reduction
GO:0009072 aromatic amino acid family metabolic process
Complex of proteins?: no
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
no chemical compounds
*EC#:1.14.16.2
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.14.16.2
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Continuous Spectrophotometric Rate Determination, 100 mM Tris HCl Buffer, pH 7.0 at 37°C (Buffer), 30 mM L-Tyrosine Solution, pH 7.0 at 37°C (L-Tyr)
-- link to Sigma (or other company) page for assay (see Sigma links below)
http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/assay-library/ec-number.html
-- links to assay reagents (substrates) pages.
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/General_Information/tyrosine_hydroxylase.pdf
--- List cost and quantity of substrate reagents, supplier, and catalog #
6,7-Dimethyl-5,6,7,8-Tetrahydropterine 100 mg in glass bottle for $105.00.
1l Tris HCL $27.00, L-Tyrosine Solution 50g for $65.50
Structure Available (PDB or Homology model)
PDB # 2MDA
Expression Information (has it been expressed in bacterial cells): yes in escherichia coli
Purification Method: one of many purification methods is chromatography
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
*length of your protein in Amino Acids: 527
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 58392.9
Molar Extinction coefficient of your protein at 280 nm wavelength: 46215 M^-1 cm^-1
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
*GC% Content for gene: 66.05%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized): 50.3%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**
Resources:
See ProtocolTargetDiscoveryVDS.docx for more
Etiologic Risk Group Categories (for pathogens): http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
SIGMA-ALDRICH RESOURCES
Enzyme Explorer
http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer.html
Enzyme Classification Index (EC number)
http://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?TablePage=14573088
WolframAlpha http://www.wolframalpha.com/
DrugBank http://www.drugbank.ca/
Databases of genes/organisms:
http://www.niaid.nih.gov/Pages/default.aspx
http://eupathdb.org/eupathdb/
https://patricbrc.vbi.vt.edu/portal/portal/patric/Home
http://www.nmpdr.org/FIG/wiki/view.cgi/Main/EssentialGenes
http://tubic.tju.edu.cn/deg/
http://csgid.org/csgid/cake/pages/community_request_gateway
http://tdrtargets.org/
http://gsc.jcvi.org/status.shtml
Scientific Nomenclature page from Center for Disease Control (gene, protein names and abbreviations)
http://wwwnc.cdc.gov/eid/pages/scientific-nomenclature.htm
Gene Information:
NCBI GENE Page: http://www.ncbi.nlm.nih.gov/gene
BLAST Page: http://blast.ncbi.nlm.nih.gov/
Protein Information:
NCBI Protein Page: http://www.ncbi.nlm.nih.gov/protein
Protein Expression Website
Protein Expression Paper: SGC_ProteinProductionPurificationNatMethods2008.pdf
Primer Overlap PCR Articles
HooverLubkowski_PCRoverlapcloninggnf042.pdf
StemmerPCRoverlapGene1995.pdf
Is my target good for Virtual Screening programs?
Reynolds_THermodynamicsLigandBinding_MedChemLett2011.pdf